Cyclic peptides are conformationally restricted and, as such, exhibit increased specificity and affinity in binding to other molecules, as compared to linear peptides. Further, cyclic peptides are thought to be more stable in cells and on the shelf than linear peptides, and may be small enough to avoid recognition by host immune system and to cross the plasma membrane of a cell (Schreiber, 2000 Science 287, 1964-1969; Scott et al., 2001 Chem. Biol. 8, 801-815).
These features make cyclic peptides very attractive drugs. Accordingly, there is a great need for new methods for making cyclic peptides, particularly for the manufacture of synthetic cyclic peptides for clinical investigations and therapeutic use, and for the production of cyclic peptide libraries that can be screened to identify cyclic peptides with a desired activity.
Current methods for making cyclic peptides, however, generally fail to meet this need. For example, linear peptides may be cyclized in vitro by reacting the N- and C-termini of a peptide together to form a covalent bond, e.g., a peptide bond, therebetween. Such methods are typically inefficient because the ends of a peptide are sterically prevented from reacting. This problem is particularly exacerbated in cyclizing smaller peptides, where the ends of the peptide have less choice of conformational space. Further, cyclic peptides made by cyclizing linear peptides can be difficult to purify from the linear peptides, and, as such, such methods sometimes require sophisticated purification procedures. Accordingly, it is often difficult to produce and purify a cyclic peptide in any useful amount using synthetic chemistry.
Further, while inteins have been used to cyclize peptides in vitro and in vivo, those peptides are typically synthesized in vivo, i.e., by ribosomes in a cell. Such cyclic peptides therefore typically contain only genetically-encodable amino acids (i.e., L-amino acids) and, as such, are limited in their structural diversity. Further, before their use, cyclic peptides produced in a cell are typically purified away from other cellular components before use. Since this is not a trivial task, cyclic peptides made by cells cannot generally be produced in large amounts, are typically not amenable to typical high throughput, cell-free, screening assays, and may not be suitable for many clinical studies. In addition, cell-based methods are limited to production of cyclic polymers that are genetically encodable.
Accordingly, there is still a great need for new methods for producing cyclic peptides. In particular, there is a great need for cell-free system methods of producing small cyclic peptides containing non-genetically encodable amino acids.
This invention meets this need, and others.